Hematopoietic stem cells are primitive, uncommitted progenitor cells that give rise to the lymphoid, myeloid and erythroid lineages of cells in blood. The stem cell population constitutes only a small proportion of the total cells in bone marrow and represents even a far more minuscule proportion of the cells in peripheral blood.
Stem cells have commonly been characterized by their surface antigenic determinants. Tsukamoto et al., U.S. Pat. No. 5,061,620 (1991), teaches that a highly stem cell concentrated cell composition is CD34+, CD10−, CD19− and CD33−. Leon et al., Blood 77:1218-1227 (1991), teaches that about one per cent of CD34+ cells, or about 0.01% of the total marrow cell population, do not express differentiation antigens, such as CD33 (myeloid lineage), CD71 (erythroid lineage) or CD10 and CD5 (lymphoid B and T lineage), and that reduced expression of CD34 expression during maturation is associated with increased expression of the differentiation antigens.
Combinations of antigenic and functional characteristics have also been used to characterize stem cells. Sutherland et al., Proc. Natl. Acad. Sci. U.S.A. 87:3584-3588 (1990), teaches that primitive stem cells do not bind to soybean agglutinin, express high levels of CD34, form blast colonies with high plating efficiency and are enriched in long-term culture initiating cells (LTC-IC). Craig et al., Blood Reviews 6:59-67 (1992), teaches that the CFU-GM assay is the most widely used measure of the hematopoietic progenitor viability of a bone marrow or peripheral blood stem cell harvest, and correlates well with per cent CD34+. Spangrude, Immunology Today 10:344-350 (1989), teaches that stem cells accumulate low levels of rhodamine 123 relative to other bone marrow cell types. Chaudhaury et al., Cell 66:85-94 (1991), teaches that stem cells express high levels of P-glycoprotein relative to other marrow cell types.
The ability to manipulate hematopoietic stem cells has become increasingly important in the development of effective chemotherapeutic and radiotherapeutic approaches to the treatment of cancer. Current approaches to chemotherapy and radiotherapy utilize bone marrow transplantation (BMT). BMT involves removing one to two liters of viable pelvic bone marrow containing stem cells, progenitor cells and more mature blood cells, treating the patient with chemotherapy or radiotherapy to kill tumor cells, and reintroducing bone marrow cells intravenously. BMT, however, suffers from many disadvantages. Harvesting of BM for BMT requires general anaesthesia, which increases both risk and cost. In addition, if cancer cells are present in the marrow and are not rigorously purged, recurrence of the disease is a distinct risk. Also, if widespread invasion of bone marrow by cancer cells (myeloma, Waldenstrom's macroglobulinemia) is present, peripheral blood cells are the only option for use in autologous transplantation (ABMT). Finally, patients who have undergone pelvic irradiation are not candidates for ABMT.
As a result of these difficulties, much interest has been developed in providing methods for obtaining stem cells from peripheral blood for autologous supply of stem cells to patients undergoing chemotherapy. Autologous supply of stem cells from peripheral blood would allow the use of greater doses of chemo- or radiotherapy, but with less risk than BMT. In addition, the use of stem cells from peripheral blood does not require anaesthesia to obtain the stem cells. Also, Lowry, Exp. Hematol. 20:937-942 (1992), teaches that cancer cells in the marrow tend not to peripheralize. The critical limitation in such a procedure, however, lies in the very small number of stem cells ordinarily present in peripheral blood. Lobo et al., Bone Marrow Transplantation 8:389-392 (1991), teaches that addition of peripheral blood stem cells collected in the absence of any peripheralization techniques does not hasten marrow recovery following myeloablative therapy. In contrast, Haas et al., Exp. Hematol. 18:94-98 (1990), demonstrates successful autologous transplantation of peripheral blood stem cells mobilized with recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF). Thus, increasing the number of stem cells in peripheral blood by peripheralization techniques is critical to the success of procedures utilizing peripheral blood as a source for autologous stem cell transplantation. Other cytokines may be useful in this regard. Rowe and Rapoport, J. Clin. Pharmacol. 32:486-501 (1992), suggests that in addition to GM-CSF, other cytokines, including macrophage colony-stimulating factor (M-CSF), granulocyte colony-stimulating factor (G-CSF), erythropoietin, interleukins-1, -2, -3, -4 and -6, and various interferons and tumor necrosis factors have enormous potential.
Another approach to autologous transplantation is to purify stem cells from peripheral blood using immunoaffinity techniques. These techniques hold promise not only for autologous stem cell transplantation in conjunction with chemotherapy, but also for gene therapy, in which purified stem cells are necessary for genetic manipulation to correct defective gene function, then reintroduced into the patient to supply the missing function. However, Edgington, Biotechnology 10:1099-1106 (1992), teaches that current procedures require three separate four hour sessions to process enough cells in the absence of peripheralization. DePalma, Genetic Engineering News, Vol. 12, May 1, 1992, teaches that this can be improved by treatment with G-CSF for peripheralization.
These studies underscore the importance of developing new methods to effect the peripheralization of hematopoietic stem cells. One possibility is to search for new ways to release stem cells from the bone marrow environment into the periphery. Unfortunately, little is known about the types of molecular interactions that hold hematopoietic stem cells in the marrow environment in vivo. Recently, some in vitro studies have been undertaken to look at the role of integrins, fibronectin, and other surface antigens in binding between stem cells and bone marrow stromal cells.
Integrins are a large family of integral membrane glycoproteins having over 16 heterodimeric members that mediate interactions between cells, interactions between cells and the extracellular matrix, and interactions involved in embryonic development and regulation of T-cell responses. Among integrins, the VLA-5 (α5β1) complex is widely distributed and functions as a receptor for fibronectin. The VLA-4 (α4β1) complex is expressed at substantial levels on normal peripheral blood B and T cells, thymocytes, monocytes, and some melanoma cells an well as on marrow blast cells and erythroblasts. Ligands for VLA-4 are vascular cell adhesion molecule-1(VCAM-1) and CS-1, an alternately spliced domain within the Hep II region of fibronectin. Another group of integrins (CDIIa/CD18, CDIIb/CD18, and CDIIc/CD18) share the common β2 chain and are variably expressed on peripheral T cells, monocytes, and mature granulocytes. Ligands for β2-integrins include members of the Ig superfamily (ICAM-1 and ICAM-2) found on activated endothelial cells.
Issekutz, J. Immunol. 147:4178-4184 (1991), discloses that TA-2, a monoclonal antibody to rat VLA-4, inhibits the in vivo migration, of small peritoneal exudate lymphocytes and lymphocytes from peripheral lymph nodes, from the blood across the vascular endothelium to sites of inflammation. This document also observes that systemic treatment of rats with TA-2 was accompanied by an increase in total blood lymphocyte count.
Taixido et al., J. Clin. Invest. 90:358-367 (1992), teaches that in an in vitro model, interactions between VLA-4/VCAM-1, VLA-5/fibronectin and β2-integrin/ICAM-1 are all important for adhesion between bone marrow stromal cells and cells expressing high levels of CD34. Simmons et al., Blood 80:389-395 (1992), teaches that in an in vitro model, adhesion between stromal cells and CD34− cells was predominantly dependent on the VLA-4/VCAM-1 interaction and was largely inhibited by monoclonal antibodies to either VLA-4 or VCAM-1, with fibronectin playing a minor role in binding. Williams et al., Nature 352:438-441 (1991), using in vivo mouse studies, teaches that adhesion of murine hematopoietic stem cells to stromal cell extracellular matrix (ECM) is partly promoted by proteolytic fragments of fibronectin containing an alternatively spliced region of the IIICS domain, and suggest that the interaction is likely to be mediated by VLA-4. All of these studies utilized antibodies to prevent adherence between stem cells and their microenvironment. However, none have analyzed whether such interactions are reversible, or perturbable after adherence has taken place. These results indicate the need for further studies to determine what interactions between the bone marrow environment and hematopoietic stem cells are responsible for keeping the stem cells within that environment in vivo and whether such interactions can be perturbed to effect peripheralization of stem cells.
There is, therefore, a need for new methods for peripheralizing stem cells, both for scientific investigatory purposes for understanding the processes of peripheralization and homing, and for the development of better methods of peripheralization for autologous stem cell transplantation in the course of cancer treatment or gene therapy. Preferably, such methods should produce even higher levels of stem cells in peripheral blood than existing methods provide.